The major premise of this proposal is that endothelial regeneration is critical for cardiovascular health, and that therapeutic strategies to enhance this process will reduce cardiovascular disease. This proposal is designed to provide fundamental insights into the process of endothelial regeneration, with specific attention to its role in angiogenesis. Moreover, this work will provide the foundation for our group to transfer these basic insights into stem cell therapy for peripheral arterial disease. We and others have shown that NO plays a critical role in the survival, proliferation, migration, and capillary formation of endothelial cells. Accordingly, the focus of our first specific aim is to develop technologies for the application of embryonic stem cell (ESC) therapy in a model of peripheral arterial disease and to assess the efficacy of this technology. We will characterize the differentiation, proliferation, and incorporation of embryonic stem cell (ESC)-derived endothelial cells (EC) into the vasculature. (a) We will generate an ESC line optimized for purification ex vivo and non-invasive tracking in vivo, (b) We will determine if ESC-derived EC, infused into the systemic vasculature, can incorporate into sites of endothelial repair, and have a functional effect. The second specific aim examines the role of nitric oxide synthase (NOS) in the differentiation, proliferation and incorporation of ESC-derived endothelial cells (EC) into the vasculature. (a) We will determine if alterations in ADMA have a paracrine influence on the homing, incorporation into the vasculature, and proliferation of ESC-derived EC. (b) We will determine the mechanisms by which ADMA influences ESC differentiation and proliferation. This proposal will provide fundamental insights regarding the use of ESC- derived EC for endothelial regeneration, with specific attention to therapeutic angiogenesis, and its regulation by NOS. [unreadable] [unreadable] [unreadable]